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The Journal of Immunology, 1998, 161: 3393-3399.
Copyright © 1998 by The American Association of Immunologists

{gamma}3 Gene-Disrupted Mice Selectively Deficient in the Dominant IgG Subclass Made to Bacterial Polysaccharides Undergo Normal Isotype Switching After Immunization with Polysaccharide-Protein Conjugate Vaccines1

David A. Shapiro*, Deborah S. Threadgill3, M. Janna Copfer*, Deborah A. Corey*, Tera L. McCool*, Laura L. McCormick{ddagger}, Terry R. Magnuson{dagger}, Neil S. Greenspan2,{ddagger} and John R. Schreiber2,4,*

Departments of * Pediatrics and {dagger} Genetics, and {ddagger} Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the {gamma}3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3-/- mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3-/- mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3-/- mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream {gamma}3 heavy-chain gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial polysaccharides (PS)5 are "T-independent type 2" (TI-2) Ags that elicit Abs of restricted clonotype and isotype (IgM and IgG3 in mice, IgM and predominantly IgG2 in humans) late in ontogeny in mammals (1, 2, 3). The importance of the dominant IgG subclass Ab elicited by PS to host defense against bacterial infection is unclear, although humans deficient in IgG2 often have chronic sinopulmonary infections with PS-encapsulated bacteria (4). Mouse IgG3 is immunobiologically similar to human IgG2 in that it is the major IgG subclass made to most T-independent Ags and is made late in mouse ontogeny (5, 6). Thus, neonatal mice make little IgG3 (or other isotypes) in response to immunization with pure bacterial PS. Human infants (<2 yr old) exhibit a similar unresponsiveness to PS. Interestingly, when both mice and humans are immunized with PS conjugated to proteins, which is a method used clinically to enhance the immunogenicity of bacterial PS, the dominant IgG subclass that is made is IgG1 (7, 8, 9).

Murine IgG3 has been shown previously to exhibit PS binding superior to that of other IgG subclasses due to noncovalent cooperativity between IgG3 Fc regions that endows this isotype with an enhanced "functional affinity" over other isotypes (such as IgG1) for binding to multivalent PS (10, 11). Similarly, IgG3 directed against Pseudomonas aeruginosa LPS O-side chain exhibited superior Ag binding and opsonophagocytic capacity compared with a variable region-identical monoclonal IgG1 isotype-switch variant (12). IgG3 also proved better than other isotypes at protecting mice from fatal pneumococcal infection (13). In contrast, IgG3 directed against cryptococcal capsular epitopes has been found to be detrimental to host defense against the organism (14). Upon switching the isotype of this Ab to IgG1, however, protective efficacy of passive Ab administration was demonstrated. Thus, the precise role of the dominant IgG subclass made to PS epitopes in host defense against encapsulated pathogens may differ depending upon the organism.

To elucidate the role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mutant mice with a targeted disruption of the exon encoding the CH1 domain of the {gamma}3 heavy-chain constant region gene. This directed mutation model, which is completely deficient in IgG3, will aid in the determination of the biologic importance of IgG3 produced in response to immunization with bacterial PS and will help clarify the role of this dominant anti-PS subclass in host defense against a variety of encapsulated pathogens. In addition, the specific disruption of the {gamma}3 gene provides insight into the mechanism of isotype switching that occurs from IgG3 to IgG1 when PS are conjugated to carrier proteins, a procedure used to enhance the immunogenicity of bacterial PS vaccines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Mice were housed in microisolator cages in the Animal Resource Center at Case Western Reserve University School of Medicine; all animal protocols were approved by the Animal Care and Use Committee of Case Western Reserve University. Animals were fed a diet of autoclaved Teklad mouse chow (Harlan Sprague-Dawley, Troy, IL) ad libidum. Sera from naive and immunized animals were prepared from 100-µl whole blood samples drawn weekly via the tail vein.

Targeting vector

A {lambda}129SV genomic library (Stratagene, La Jolla, CA) was screened using an 800-bp {gamma}3 sequence-specific probe. One {lambda} clone was obtained from this screen. A neomycin-resistance cassette bearing a ß-actin promoter was then introduced into a plasmid vector containing a 6.8-kb HindIII fragment of the {gamma}3 heavy-chain constant region locus such that 54 bp of {gamma}3 CH1 were deleted (Fig. 1GoA) leaving the switch region intact. For selection against nonhomologous recombination events, a diphtheria toxin gene was attached to the 5' SpeI site of the {gamma}3 sequence (15). This construct was cloned into pBluescript SK+ (Stratagene) and linearized with HindIII (p{gamma}3TV). The targeting vector was introduced into E14-1 embryonic stem cells (16, 17) (a gift of Dr. C. Colmenares, Cleveland Clinic Foundation, Cleveland, OH) via electroporation. Cells were plated and grown in a DMEM-based selective medium containing G418. Embryonic stem cells containing the targeted construct were injected into blastocysts obtained after the fertilization of C57BL/6 female mice with C57BL/6 males; the injected blastocysts were then transferred to the uteri of pseudopregnant F1 C3HB16 females. Resulting males (agouti/C57BL) expressing a chimeric coat color (>25% agouti/chinchilla agouti color) were bred to National Institutes of Health Swiss Black mates. Germline chimeras were defined as producing progeny with an agouti coat color. Male and female agouti littermates (heterozygous agouti/C57BL/Swiss) were then crossed to obtain homozygous knockout (KO) pups. Homozygous wild-type (WT) littermates were saved and bred as controls.



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  		FIGURE 1. Gene targeting of the murine {gamma}3 heavy-chain locus. A, Schematic representations, from top to bottom, of the p{gamma}3 targeting vector, genomic DNA, and the mutant {gamma}3 locus. B, Southern blot analysis of embryonic stem cell DNA using the 400-bp probe. HindIII-digested genomic DNA of transfectants and the parental cell line (E14-1) were hybridized with the probe. E14-1 cells show the native 6.7-kb band, while a successful transfectant (+/-) has both the 6.7-kb band and an 8.9-kb band (reflecting the size of the fragment with insert) after digestion with HindIII. C, Southern blot analysis of HindIII-digested genomic tail DNA of WT (+/+), heterozygous (+/-), and homozygous KO (-/-) offspring from the mating of heterozygous pups from a chimeric founder crossed with a C57BL/6 mate.

 
Tail DNA Southern blots

Genomic DNA from mouse tail clippings was digested overnight using 10 U of HindIII, loaded onto 1% agarose gels, and electrophoresed for 375 V/h. Following denaturation for 2 h in 0.5N NaOH containing 1.5 M NaCl (with one change after 30 min), DNA was blotted overnight via capillary transfer to Nytran (Schleicher and Schuell, Keene, NH) in 20x SSC buffer (18). The blot was washed, UV cross-linked (1200 x 100 µJ/cm2), and prehybridized with Church buffer (19) at 65°C for 1 h. Next, the blot was hybridized overnight at 65°C with a 400-bp 32P-labeled homologous probe (prepared by random primer labeling) and visualized using a PhosphorImager (model 400S, Molecular Dynamics, Sunnyvale, CA).

Isotype ELISA

96-well microtiter plates (Corning Plasticware, Corning, NY) were coated with 0.1 µg/well of anti-murine Ig (Southern Biotechnology Associates (SBA), Birmingham, Alabama) overnight at 4°C and blocked with 240 µl/well 1% BSA (Sigma, St. Louis, MO) in PBS (50 mM phosphate and 150 mM NaCl (pH 7.2)) for 1 h. Blocked wells were incubated overnight with serial dilutions of sera from unimmunized WT and IgG3-/- mice diluted in 1% BSA/PBS. Bound Igs were detected by the addition of a 1/1000 dilution of isotype-specific alkaline phosphatase (AP)-conjugated goat polyclonal Abs (SBA) (100 µl/well in 1% BSA/PBS) for 1 h at room temperature followed by development with 100 µl/well of p-nitrophenyl phosphate (pNPP) chromogenic substrate (Sigma). Absorbance was determined spectrophotometrically at 410 nm using a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Purified murine Igs (SBA) served as control proteins. Group mean values for this assay and the following isotype titration assays were compared by the Student t test using the StatView software package (Abacus Concepts, Berkeley, CA) for Macintosh. Differences were considered significant when p values were <=0.05.

Splenocyte cultures

Spleens from naive WT or IgG3-/- mice were aseptically removed into RPMI 1640 (Life Technologies, Grand Island, NY). Single-cell suspensions were prepared by grating the spleens over a wire mesh, and RBCs were removed by incubation with an ammonium chloride lysis buffer (155 mM ammonium chloride and 17 mM Trizma (pH 7.2)). Cells at 5 x 106/ml were distributed into the wells of 12-well microtiter plates (Corning) and incubated for 4 days in RPMI 1640-based complete medium containing 10% FBS with either 30 µg/ml Escherichia coli LPS (serotype O127:B8, Sigma) or media without LPS (control) (20). Cells were harvested and counted, and supernatants were saved for analysis of secreted Igs. Supernatants were analyzed using the isotype ELISA described above.

Anti-P. aeruginosa PS ELISA

The high m.w. component from the O-side chain of Fisher-Devlin immunotype 1 P. aeruginosa LPS (high m.w. PS, kindly supplied by Dr. Gerald Pier, Channing Laboratories, Harvard Medical School, Cambridge, MA) (21, 22) was tyraminated for improved binding to polystyrene plates (23) and coated onto 96-well microtiter plates (Corning) at 4 µg/ml in PBS. Unbound sites were blocked with 1% BSA/PBS. Serial dilutions of sera (weekly bleeds) from mice immunized i.p. with 10 µg of the same (nontyraminated) PS in a total volume of 100 µl 1% BSA/PBS were incubated in duplicate wells overnight at 4°C. Bound Ab was detected by the addition of subclass-specific AP-conjugated polyclonal goat Abs (100 µl/well of a 1/1000 dilution in 1% BSA/PBS for 1 h at room temperature; SBA) followed by the addition of 100 µl/well of pNPP chromogenic substrate (Sigma). Absorbance was determined spectrophotometrically at 410 nm. Statistical differences were determined as in the isotype ELISA described above.

Anti-Streptococcus pneumoniae PS ELISA

96-well microtiter plates (Corning) were coated with PS from the capsule of type 19F S. pneumoniae (#6319, Advanced Type Culture Collection, Atlanta, GA) at 10 µg/ml, 100 µl/well overnight at 4°C in PBS. Unbound sites were blocked with 1% BSA/PBS. Cross-reactive anti-cell wall PS serum components were removed by adsorption of sera with 50 µg S. pneumoniae cell wall PS (University of Rochester, Rochester, NY) per milliliter of serum for 1 h at 4°C (24). Mice were immunized i.p. with 10 µg in 100 µl PBS of the 19F PS or with 19F covalently linked to cross-reacting material (CRM)197, a nontoxic mutant of diphtheria toxin (25) that was obtained from Lederle-Praxis Biologicals (Rochester, NY), and conjugated as described previously (26). Serial dilutions of sera taken from weekly bleeds were incubated in 1% BSA/PBS in duplicate wells at 100 µl/well overnight at 4°C. Bound anti-PS was detected by the addition of subclass-specific AP-conjugated polyclonal Abs (diluted 1/1000 in 1% BSA/PBS; SBA) followed by the addition of 100 µl of pNPP chromogenic substrate (Sigma). Absorbance was determined at 410 nm.

Cell preparation and flow cytometric analysis of surface IgM

Spleens from naive mice or mice immunized with immunotype 1 P. aeruginosa high m.w. PS were removed into PBS. Single-cell suspensions were prepared by grating the spleens over a wire mesh. RBCs were removed by incubation with an ammonium chloride lysis buffer. Tubes containing 106 cells were incubated first with a nonspecific anti-Fc{gamma} block and then with fluorophore-labeled Abs (or fluorophore-labeled isotype-matched controls). All cells were stained with both a B cell-specific stain (phycoerythrin-anti-B220) and a surface IgM-specific FITC-labeled Ab. Flow cytometry was performed on a Becton Dickinson FACScan (Mountain View, CA) using Lysis II analysis software. A total of 10,000 B220+ cells were gated and analyzed from each sample.

Statistics

Serum Ig concentration group means were compared using the Student t test. Where variances between groups were unequal, the Welch test was used, establishing an average variance with which to determine t values. p values were deemed significant if they were <0.05. Error bars in all figures represent SEMs. All group statistics were calculated using the StatView statistical package for Apple Power Macintosh computers (Cupertino, CA). Kolmogorov-Smirnov statistics were used to compare the equivalence of flow cytometric fluorescence data using PC-Lysis software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of IgG3-deficient mice

The murine {gamma}3 gene was targeted for disruption via homologous recombination in embryonic stem cells. A total of 54 bp of CH1 were replaced in one {gamma}3 allele in murine embryonic stem cells with a targeting vector containing the diphtheria toxin gene and a neomycin-resistance cassette (neor) bearing a ß-actin promoter (Fig. 1GoA). Successful transfectants (Fig. 1GoB) were used to make blastocyst injection chimeras. Germline transmission of the mutated {gamma}3 allele was then demonstrated in the progeny of these chimeras by Southern blotting of tail DNA (Fig. 1GoC).

Absence of detectable IgG3 in KO mice

Mice homozygous for the mutated allele (IgG3-/-) (Igh-8tm1Sch) had no circulating IgG3 as measured by ELISA (Fig. 2GoA). Levels of total serum Ig and all other IgG subclasses were statistically similar to those in WT control mice (Fig. 2GoB). However, IgM levels in naive IgG3-/- mice were significantly elevated relative to IgM levels in IgG3+/+ mice (Fig. 2GoB; p < 0.005). Flow cytometry on splenic lymphocytes (Fig. 2GoC) revealed no significant difference in the percentage of B cells expressing surface IgM between IgG3+/+ and IgG3-/- mice (Kolmogorov-Smirnov analysis). Interestingly, a similar increase in IgM anti-PS levels in IgG3-/- mice compared with controls was observed upon immunization with a PS Ag commonly known to elicit IgG3 Abs following early IgM production, as shown in Figure 4GoA and discussed below.



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  		FIGURE 2. Detection of soluble IgG3. A, Serum IgG3 concentrations in naive IgG3-/- mice (KO; open symbols) and IgG3+/+ mice (WT; closed symbols) as measured by solid-phase ELISA. IgG3 is clearly present in detectable quantities in the sera of resting WT mice and is completely undetectable in the sera of IgG3-/- mice. B, Serum Ig concentrations for each isotype in naive (unimmunized) mice. IgG3 concentrations in mutant mice were below the level of detection in this assay. Interestingly, IgG3-/- mice appeared to have resting IgM levels in excess of their WT siblings. C, Quantitation of surface IgM in naive IgG3-/- and WT splenocytes by flow cytometry. There was no significant difference in the percentage of B220+ cells expressing surface IgM between IgG3+/+ and IgG3-/- mice (89.4% vs 86%). In addition, mean fluorescence per cell (arbitrary units) was similar in both populations (1027 in IgG+/+ cells vs 1105 in IgG3-/- cells). The differences in the fluorescence profiles for the two cell populations were not statistically significant by Kolmogorov-Smirnov analysis.

 


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  		FIGURE 4. Serum Ab titrations upon immunization with pure PS or with PS conjugated to a protein carrier (CRM197, a nontoxic diphtheria toxin mutant). A, Titration of serum Abs in IgG3-/- and WT mice produced in response to immunization with high m.w. PS from Fisher-Devlin immunotype 1 P. aeruginosa LPS O-side chain (high m.w. PS; a TI-2 Ag). B, Isotype distribution of serum Abs in IgG3-/- (KO) and IgG3+/+ (WT) mice in response to immunization with S. pneumoniae type 19F capsular PS. C, Isotype distribution of serum Abs in IgG3-/- (KO) and IgG3+/+ (WT) mice in response to immunization with S. pneumoniae type 19F capsular PS conjugated to the protein carrier CRM197. Unlike pure bacterial PS, the protein-conjugated 19F-CRM197 elicited an IgG response consisting primarily of the IgG1 subclass. IgG3-/- mice, despite having disrupted {gamma}3 CH1 loci, are able to generate a functional IgG1 anti-PS Ab response indistinguishable from that generated in WT mice.

 
Ig levels in LPS-treated KO and WT splenocytes

To determine whether spleen cells from IgG3-/- mice could be induced to express IgG3 in vitro, spleen cells from mutant and WT mice were harvested and incubated in the presence of LPS from E. coli for 4 days (20). Total Ig levels in both IgG3-/- and IgG3+/+ mice increased ~100-fold as a result of 4 days of polyclonal stimulation (Fig. 3Go). IgM production in both WT and mutant cell populations increased in proportion to total Ig levels, suggesting that much of the increased Ig output was due to elaboration of IgM under in vitro conditions. Supernatant IgG3 production in WT cells increased from 18 to 300 µg/ml following LPS exposure. Splenocytes from IgG3-/- mice did not make detectable supernatant IgG3 Ab either with or without LPS stimulation.



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  		FIGURE 3. In vitro Ig isotype titers in LPS-stimulated lymphocyte cultures. Supernatant titers for three representative isotypes (IgM, IgG3, and IgG1) were compared in splenocytes from IgG3-/- (KO) and IgG3+/+ (WT) mice. LPS effectively stimulated 10- to 100-fold increases in supernatant Ig isotype levels in WT mice and in mutant mice in all but the IgG3 subclass.

 
Anti-Pseudomonas high m.w. PS Ab production

Immunizing BALB/c mice with the endotoxin-free high m.w. PS component of the O-side chain of P. aeruginosa LPS has been shown previously to elicit IgM and IgG3 Abs in a classical TI-2 response (21, 22). After i.p. immunization with this PS Ag, IgG3-/- mice generated a higher IgM Ab titer within the first week after immunization than did WT mice (Fig. 4GoA). By contrast, WT mice elaborated predominantly IgM and IgG3 Abs, slowly increasing in titer after the first week. Flow cytometric analysis of WT mice B cell surface IgM before and after PS immunization showed no difference in the percentage of B cells expressing surface IgM at 2 wk postimmunization with high m.w. PS (92.2% in nonimmunized vs 92.9% in immunized mice; data not shown).

Isotype switching upon immunization with PS-protein conjugate vaccine

To answer the question of whether PS-protein conjugate-driven anti-PS Ab isotype switching to IgG1 can occur in the absence of a functional {gamma}3 heavy-chain gene, mutant and WT mice were immunized with the T-independent capsular PS from S. pneumoniae type 19F or with the same PS Ag conjugated to a nontoxic mutant of diphtheria toxin (CRM197). This carrier protein is known to induce an Ab response resembling that elicited by T-dependent Ags when linked to bacterial PS (27). In response to pure PS, WT mice produced IgM and low levels of IgG3 anti-PS Abs (Fig. 4GoB). In contrast, the mutant mice were unable to generate IgG3 anti-PS Abs, producing almost exclusively IgM anti-PS. When challenged with a "T cell-dependent" form of the PS (conjugated to the protein carrier CRM197), however, both WT and mutant mice responded by producing high titers of IgG1 anti-PS Abs (Fig. 4GoC). There were no detectable IgA, IgG2a, or IgG2b Abs elicited by either the PS or the conjugate vaccine. These results were reproducible in repeat experiments employing different lots of conjugate vaccine (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous mouse models of hyporesponsiveness to T-independent Ags have involved mutations yielding complex phenotypes. These phenotypes exhibit numerous alterations in addition to those affecting Ab isotype. One such model is the X-linked immunodeficiency disorder (xid), which has been characterized by abnormally low levels of serum IgG3 and IgM (28). The xid phenotype has a general unresponsiveness to TI-2 Ags, including bacterial PS, stemming from a defect in the gene for Bruton’s tyrosine kinase (29, 30, 31, 32). B cells from xid mice have a high rate of apoptosis (33) and do not proliferate upon cross-linking of surface Ig (34). This phenomenon was recently shown to be a function of aberrant cell cycle entry of the xid B cells, which is characterized by a lack of cyclin induction following the progression from G0 to G1 (35). Although IgG3 hyporesponsiveness is a characteristic of the xid phenotype, the inherent complexity of the interactions between the kinase and its substrates precludes one from using this model system to make conclusive determinations about isotype restriction in the host response to bacterial PS.

Similar hyporesponsiveness to T-independent Ags was obtained in a model in which mice lack most of the Ig-{alpha} tail of the B cell receptor (BCR) complex (36). These mice proved to be unresponsive to the TI-2 Ag 4-hydroxy-3-nitrophenylacetyl coupled to Ficoll, while still producing an Ab response to the T-dependent form of 4-hydroxy-3-nitrophenylacetyl coupled to chicken gammaglobulin (although this response was reduced to ~1% of that seen in controls in accordance with a similar reduction in mature splenic B cell numbers). B cell development and function in this model resembled that of the X-linked immunodeficiencies in humans and mice. In fact, since it has been suggested that the Bruton’s tyrosine kinase signal transduction pathway may originate at the BCR (37), the agammaglobulinemias exhibited in these two model systems probably reflect similar, complex immunoregulatory cascades initiated at the level of BCR signaling.

Consequently, the general hyporesponsiveness in these animal models to bacterial PS Ags is not limited to the changes in the dominant Ig subclass(es) produced in response to bacterial infection or exposure to T-independent Ags. Therefore, these animals are not ideal as models of human Ig subclass deficiency or for the study of the class-restricted response to bacterial PS. The development of a new mouse strain by targeted mutagenesis of the gene for IgG3 provides a more specific animal model of IgG subclass deficiency associated with the humoral response to TI-2 Ags.

As noted in the text, total serum IgM levels were higher in naive IgG3-/- mice than in WT controls. Possible reasons for the relatively elevated IgM levels in IgG3-/- mice include the existence of a larger pool of IgM+ B cells, a propensity for existing IgM clones to become plasma cells, differences in rates of IgM secretion between WT and KO cells, or decreased IgM catabolism in the KO phenotype. The latter two phenomena seem unlikely in this very specific directed mutation model. In addition, numbers of IgM+ B cells were measured and shown by flow cytometry to be comparable in mutant and WT littermates. Furthermore, LPS stimulation did not result in increased IgM secreted by spleen cells from mutant mice (Fig. 3Go). A more likely hypothesis for our data is that the increased IgM represents a compensatory reaction to stimulation by environmental Ags in the absence of the ability to manufacture IgG3. Similarly, PS-specific IgM was elevated in mutants compared with controls after immunization with a TI-2 Ag (high m.w. PS). The PS-specific B cells in the {gamma}3-deficient animals were unable to make IgG3 after immunization; consequently, the increased IgM is likely the result of these B cells continuing to secrete IgM (PS-specific maturational arrest).

Recent attempts to enhance the immunogenicity of bacterial PS by linking them to protein carriers have produced vaccines exhibiting many of the properties of T-dependent Ags, including a booster response upon reexposure and isotype switching to IgG1 Abs in both mice and humans (38), presumably as a consequence of signals delivered by stimulated Th cells. The single Ig heavy-chain gene defect in IgG3-/- mice (with an intact {gamma}3 switch region) provides a unique opportunity to study the requirement for ordered progression of expression of Ig genes from 5' to 3' in the Ab response to PS and to compare this response with that generated to a PS-protein conjugate. The sequential expression of Abs of identical specificity but different isotype by B cells occurs via switch recombination, in which the variable region genes are moved in proximity to different heavy chain constant region genes; this action is currently believed to occur via recombination at specific upstream switch regions involving a looping-out deletion of intervening DNA (39, 40, 41, 42). If isotype switching were reliant on a sequential generation of functional upstream isotypes, the disruption of the {gamma}3 heavy-chain gene and the lack of IgG3 expression developed in this model would cause an inability to express downstream isotypes such as IgG1. Such immunoregulatory aspects of sequential isotype switching have, for example, been hypothesized for IgG1 in the control of IgE expression (43). However, recent data in a mutant mouse with a disrupted {gamma}1 switch region have shown that the rate of class switching to IgE is not affected by the elimination of IgG1 expression. Similarly, in the same mutant mice, downstream class switching to IgG2a appeared to be independent of the specific ability or inability to class switch to IgG1 (44, 45, 46).

Immunizing IgG3-/- mice with a PS-protein conjugate yielded anti-PS Abs with isotype distributions similar to those in WT mice (although notably lacking any IgG3 anti-PS). Thus, we have shown that although the IgG3 deficiency in this animal model does not prevent the expression of anti-PS Ig isotypes (other than IgG3), it does alter the distribution and magnitude of the isotype responses to immunization with TI-2 PS. The ability of mutant mice to successfully use the {gamma}1 heavy-chain gene downstream of the disrupted {gamma}3 gene suggests that sequential switch recombination and the expression of IgG3 is not necessary for the expression of IgG1 in vivo. Future studies using bacterial challenge in IgG3-deficient mice will allow us to elucidate the importance of the dominant IgG subclass made to PS in host defense against infection with encapsulated bacteria.


   Acknowledgments
 
We thank Dr. Clifford Harding for his critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 32596 (to J.R.S.), AI 27862 (to J.R.S.), DK 09179 (to L.L.M.), and Training Grant T32 CA 73515 (to D.A.S.). Back

2 These authors shared senior authorship. Back

3 Current address: Vanderbilt University, Department of Infectious Diseases, 1161 21st Avenue, A3310 MCN, Nashville, TN 37232. Back

4 Address correspondence and reprint requests to Dr. John R. Schreiber, Division of Infectious Diseases, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address: Back

5 Abbreviations used in this paper: PS, polysaccharide(s); WT, wild-type; TI-2, T-independent type 2; xid, X-linked immunodeficiency disorder; BCR, B cell receptor; KO, knockout; AP, alkaline phosphatase; pNPP, p-nitrophenyl phosphate; CRM, cross-reacting material. Back

Received for publication March 31, 1998. Accepted for publication June 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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